Pseudomonas aeruginosa is a gram-negative pathogen that is the major cause of hospital-acquired pneumonia. To cause serious infections, P. aeruginosa uses a type III secretion system to directly inject effector proteins into the cytoplasm of eukaryotic cells. The four effector proteins currently identified are ExoU, ExoS, ExoT, and ExoY. ExoU and ExoS have been most closely linked to severe disease in human and animal models of acute pneumonia. Interestingly, most clinical isolates secrete either ExoU or ExoS but not both, suggesting that they play functionally similar or redundant roles in pathogenesis. Understanding how these two enzymatically different toxins both allow P. aeruginosa to cause severe pneumonia will lay the foundation to develop therapeutics to prevent and treat P. aeruginosa infections. This project aims to understand the pathogenic mechanism of ExoS in pneumonia. Preliminary data show that ExoS-secreting strains persist in the lungs at high numbers whereas deletion of the gene results in rapid clearance from the lung over the initial 24 hr of infection. ExoS[+] strains are able to persist despite eliciting robust recruitment of neutrophils and macrophages to the lung, suggesting that ExoS somehow incapacitates these cells. This project will determine if ExoS acts to prevent P. aeruginosa clearance from the lungs by impairing phagocytes during early pneumonia. Using a mouse model of acute pneumonia and an optimized FRET-based reporter assay, the cell types injected with ExoS during the early stages of acute pneumonia will be identified. The effect of ExoS intoxication on phagocytes during early pneumonia will be defined by examining bacterial internalization by phagocytes, killing of bacteria by these cells, and phagocyte survival. Various in vitro and in vivo assays, including a fluorescence-based internalization assay, will be used to study this effect on phagocytes. These aims will define the cellular targets of ExoS and the consequences of intoxication with ExoS on these cells during early pneumonia. Such information, in conjunction with the current knowledge on the mechanisms of ExoU, will lead to a better understanding of the overall pathogenesis of P. aeruginosa during infection.